Listeria monocytogenes is an intracellular bacterial pathogen that causes serious foodborne illness in pregnant women, the elderly, infants and immunocompromised individuals. L. monocytogenes infections have the highest case fatality rate of all reported foodborne illnesses. L. monocytogenes can be easily cultured outside of host cells under standard laboratory conditions, making L. monocytogenes a significant public health risk and a significant potential threat as a biological weapons agent. Animal models show that protective immunity to L. monocytogenes is mediated by CD8+ effector cells that recognize and eliminate infected host cells. Vaccine studies have demonstrated that stimulation of protective CD8+ effector cells requires subclinical infection with live bacteria. However, immunization of humans with virulent bacteria fully capable of intracellular replication imposes a significant health risk to any population as a vaccine strategy, especially for those individuals inherently at risk to L. monocytogenes infection. We have recently developed a novel strategy for the generation of replication-deficient bacterial vaccine vectors that are capable of stimulating protective CD8+ effector cell responses. The focus of this proposal is to utilize this approach to produce non-replicating L. monocytogenes vaccine strains capable of generating protective CD8+ effector cell responses. In Specific Aim I, we will construct non-replicating L. monocytogenes vaccine vectors to deliver protective native bacterial antigens to the cytosol of professional and nonprofessional antigen presenting cells (APC) for endogenous processing and MHC Class I presentation. In Specific Aim II, we will determine the kinetics of antigen delivery to APC and the requirement of bacterial viability for efficient antigen delivery. In Specific Aim III, we will determine whether uptake of the vaccine constructs sensitizes APC for recognition by L. monocytogenes-specific effector cells. In Specific Aim IV, we will determine whether antigen specific effector cells are stimulated following immunization with the replication-deficient vaccine constructs, and assess stimulation of protective antilisterial immunity. Our goal is that following completion of the proposed studies, a safe and effective replication-deficient vaccine formulation will be identified that is suitable for clinical trials. It is also envisioned that these studies will provide a foundation for the development of replication-deficient vaccine vectors against other intracellular pathogens using a similar approach.